CN101790582A - Modified-immobilized enzymes of high tolerance to hydrophilic substrates in organic media - Google Patents
Modified-immobilized enzymes of high tolerance to hydrophilic substrates in organic media Download PDFInfo
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- CN101790582A CN101790582A CN200880021830A CN200880021830A CN101790582A CN 101790582 A CN101790582 A CN 101790582A CN 200880021830 A CN200880021830 A CN 200880021830A CN 200880021830 A CN200880021830 A CN 200880021830A CN 101790582 A CN101790582 A CN 101790582A
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6436—Fatty acid esters
- C12P7/649—Biodiesel, i.e. fatty acid alkyl esters
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- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Abstract
Disclosed are preparations of modified interfacial enzymes, particularly lipases and phospholipases, immobilized on a solid support, wherein the enzyme is surrounded by hydrophobic microenvironment, thereby protected from deactivation and/or aggregation in the presence of hydrophilic agents, substrates and/or reaction products. The enzyme may be protected by being covalently bonded with lipid groups which coat the enzyme, or by being immobilized or embedded in a hydrophobic solid support. Also disclosed are processes for the preparation of the hydrophobically protected enzymes. The enzymes may be efficiently used in the preparation of biodiesel.
Description
Technical field
The present invention relates to have active and stable immobilized interfacial enzymes, particularly lipase and the Phospholipid hydrolase of improvement for alcohol, especially short chain alcohol and other hydrophilic substance, and other lytic enzyme.The various industrial application and the research that the invention still further relates to the preparation method of these enzymes and these enzymes are used, particularly make usually as biofuel as the application in the lipid acid short-chain alkyl esters such as methyl esters.
Background technology
Interfacial enzymes is a class comprises two districts in their protein structure a enzyme; First district is a hydrophilic area, and second district is hydrophobic region.This unique feature makes will the preference interface zone when in a single day this class of enzymes is present in the two-phase system.Under these conditions, formed activity conformation, wherein the hydrophilic area of enzyme molecule towards water layer and hydrophobic region towards hydrophobic layer.
Lipase and Phospholipid hydrolase are modal interfacial enzymes, and in a single day it be present in just expresses its catalytic activity in the interface system.Lipase (triglyceride hydrolysis enzyme E.C.3.1.1.3) is defined as acting on the ester bond of the triglyceride level in the water-based system to produce the lytic enzyme of free fatty acids, partial glyceride and glycerine.Phospholipid hydrolase also belongs to hydrolase, however they cut off advantageously and specifically and be present in the ester bond of the phosphatide in the water-based system, thereby produce free fatty acids, lysophospholipid, glycerophosphonolipid, phosphatidic acid and free alcohol according to the type of Phospholipid hydrolase.
Lipase and Phospholipid hydrolase are distributed widely in animal, plant and the microorganism.Past two increases the interest of the industrial application of lipase and Phospholipid hydrolase during the decade always fast.Have been found that under low water activity this quasi-enzyme catalytic they separate reaction against the current.The contrary catalytic activity of lipase and Phospholipid hydrolase has been widely used in containing the valuable compounds therefrom of ester bond and amido linkage or has contained synthetic just like other relevant chemical substance of functional groups such as hydroxyl, carboxyl and amino.Particularly, lipase and Phospholipid hydrolase have been used to reform fat, oil, wax, phosphatide and sphingolipid to obtain the functional property of new expectation, also are used for optically active compound is separated from their racemic mixture.The application of interfacial enzymes especially meaningfully disclosed herein in short-chain alkyl ester (biofuel) synthetic.
At present, surpass 40 kinds different lipase and the commercially available acquisition of Phospholipid hydrolase, yet wherein only have minority to prepare with the commercialization amount.Some have the interfacial enzymes of industrial prospect to be derived from by antarctic candida (Candida antarctica) most, fold candida (Candida rugosa), rice black root Mucor (Rhizomucor miehei), pseudomonas (Pseudomonas sp.), snow-white head mold (Rhizopusniveus), mucor javanicus (Mucor javanicus), Rhizopus oryzae (Rhizopus oryzae), aspergillus niger (Aspergillus niger), penicillium camembertii (Penicillium camembertii), Alcaligenes (Alcaligenes sp.), bulkholderia cepasea (Burkholderia sp.), dredge the thermophilic hyphomycete of continuous shape (Thermomyces lanuginosa), thickness look bacillus (Chromobacterium viscosum), Semen Fructus Chaenomelis and pancreatin.
The immobilization of enzyme is illustrated by a large amount of technology, and these technology are basically at being reduced in the shared cost of enzyme in the entire method, helping to reclaim enzyme from product and the method that makes can be moved continuously.Immobilization technology is divided according to following condition usually:
1. enzyme is as the physical adsorption on the solid carriers such as silicon-dioxide and insoluble polymer.
2. the absorption of enzyme on ion exchange resin.
Enzyme with as the covalent attachment of solid carrier materials such as epoxidation inorganic carrier or epoxy polymer carrier.
4. the embedding in the polymkeric substance of enzyme in growth.
5. the sealing of enzyme in membrane reactor or semipermeability gel.
6. crosslinked enzyme crystal (CLECS) or cross-linked enzyme aggregate (CLEAS).
All above-mentioned enzyme immobilization programs all comprise the following steps:
1. enzyme is dissolved in the buffer system suitable for pH, temperature, buffering salt type and ionic strength.
2. join solid carrier in the enzyme solution and mix for some time, be fixed on the solid carrier until the enzyme molecule.
3. filter out the solid carrier that contains immobilized enzyme.
4. wash described carrier to remove loose bonded enzyme molecule, dry then described solid carrier with suitable buffer reagent.
Interfacial enzymes mainly is a lipase, is immobilized according to above-mentioned technology.These technology provide the immobilized enzyme goods of the work transformation period that has lower composite reactive and/or lack.In order to attempt to increase the composite reactive of immobilized lipase and other interfacial enzymes, various Activiation methods have been used.These methods comprise:
With the surface functional group of enzyme with combine as hydrophobic residue such as lipid acid or polyoxyethylene glycol.
2. with the enzyme surface that is covered as tensio-active agents such as polyhydric alcohol fatty acid esters.
3. enzyme is contacted with hydrophobic carrier, described hydrophobic carrier is generally polypropylene and has used as hydrophilic solvents such as ethanol or Virahols pretreated.
4. in reaction system, add lower concentration (being usually less than 1%) as zymoexciters such as salts solution, glycerine.
Carry out for activation, stabilization and the cost benefit of the immobilized interfacial enzymes of the reverse conversion of enzymatic with commercial quantities for being used for, aforesaid method all produces satisfied result.In addition, it is reported that most of enzymes are according to the said procedure immobilization time,, or can't show their activity performance completely because some that is subjected to that described immobilization program forces retrain, or lost their very big partial synthesis activity.For example, suffered from serious challenge with polyhydric alcohol fatty acid ester lining lipase and Phospholipid hydrolase, this moment, the lipase molecule was not to be coated with activator fully; Therefore those enzyme molecules that do not contact with activator still do not have activity.
Another main drawback of lipase and Phospholipid hydrolase is it for hydrophilic substrates, particularly the low tolerance of short chain alcohol and short chain fatty acid (being lower than C4).Many researchs are observed, and can cause as short chain alcohol such as methyl alcohol and acetate and short chain fatty acid essential water molecules is spun off from the quaternary structure of these enzymes, cause these enzyme denaturations and thereby lose catalytic activity.This shortcoming has hindered lipase has been used to use the commercialization amount preparation as the fatty acid methyl ester " biofuel " of substrate of oils triglyceride level and methyl alcohol.
Therefore, an object of the present invention is to provide a kind of the high activity also stable immobilized interfacial enzymes, the particularly lipase that are used for synthetic application and novel method of Phospholipid hydrolase of obtaining.Especially meaningfully, these enzymes can be used for synthetic as the lipid acid short-chain alkyl ester of " biofuel ".
Another object of the present invention provides and has as short chain alcohol such as methyl alcohol, ethanol and glycerine with as immobilized interfacial enzymes high activity, stable of the height endurability of short chain fatty acids such as acetate.
Along with the expansion of explanation, these purposes of the present invention and other purpose will become apparent.
Summary of the invention
The present invention relates to be fixed in the interfacial enzymes of the modification on the solid carrier, wherein, described enzyme is surrounded by hydrophobic microenvironment, can avoid inactivation and/or gathering in the presence of hydrophilic agent, substrate and/or reaction product thus.
The interfacial enzymes of modification of the present invention can be protected by being coated with covalently bound lipid groups.
Described carrier can be by being adsorbed in the functional group or by can be in conjunction with described enzyme with functional group's covalent attachment.More specifically, described carrier can be organic, also can be inorganic, and preferably be selected from by as inorganic carrier such as silica-based or alumina type carrier and the group formed as organic carriers such as polymer class carriers, wherein said carrier can contain ionic group and as epoxy group(ing) or aldehyde radical isoreactivity functional group, perhaps described carrier is an ion exchange resin.
Described lipid epoxide can be selected from lipid acid, fatty acid alkyl ester, sugar fatty acid ester, medium chain and chain alkyl glucosides, phosphatide, polyethyleneglycol derivative and quaternary ammonium salt.
In another embodiment, the interfacial enzymes of modification of the present invention is protected by being fixed on to provide on the hydrophobic solid of the hydrophobic microenvironment carrier or imbed in the described carrier.
Described enzyme can be lipase, esterase or Phospholipid hydrolase.More specifically, described enzyme can be antarctic candida (Candida antarctica), fold candida (Candida rugosa), rice black root Mucor (Rhizomucor miehei), pseudomonas (Pseudomonas sp.), snow-white head mold (Rhizopus niveus), rice black wool mould (Mucor miehei), mucor javanicus (Mucor javanicus), Rhizopus oryzae (Rhizopus oryzae), aspergillus niger (Aspergillus niger), penicillium camembertii (Penicillium camembertii), Alcaligenes (Alcaligenes sp.), bulkholderia cepasea (Burkholderia sp.), dredge the thermophilic hyphomycete of continuous shape (Thermomyces lanuginosa), thickness look bacillus (Chromobacterium viscosum), any in Semen Fructus Chaenomelis and the pancreatin.
On the other hand, the present invention relates to prepare the method for the interfacial enzymes that is fixed in the modification on the insoluble carrier, described enzyme can be avoided inactivation and/or gathering in the presence of hydrophilic agent, substrate and/or reaction product, and described method comprises the following steps:
(a) provide the system of forming by aqueous buffer and at least a organic solvent that contains the lipid epoxide;
(b) described interfacial enzymes is mixed with the solvent pairs system that step (a) is provided;
(c) described carrier is joined in the mixture of step (b) and mix;
(d) from the mixture that step (c) is obtained, isolate the interfacial enzymes that is fixed on the described carrier.
In one embodiment, described carrier is a porous support, it can be an organic or inorganic, preferably be selected from the group of forming by porous inorganic carrier (as silica-based or alumina type carrier) and organic carrier (as the polymer class carrier), wherein said carrier can contain alternatively just like epoxy group(ing) or aldehyde radical isoreactivity functional group, perhaps ionic group.
In another embodiment, described organic solvent is selected from alkanes (as octane), ethers (as diisopropyl ether), alcohols (as n-Octanol), aldehydes (as capraldehyde) and ketone (as methyln-hexyl ketone) and their any mixture.
In another embodiment, described lipid epoxide is selected from lipid acid, fatty acid methyl ester, sugar fatty acid ester, medium chain and chain alkyl glucosides, phosphatide, polyethyleneglycol derivative and quaternary ammonium salt.
On the other hand, the invention provides preparation and be fixed in the method for the interfacial enzymes of the modification on the solid carrier, wherein, described enzyme is surrounded by hydrophobic microenvironment, can avoid inactivation and/or gathering in the presence of hydrophilic substrates and/or reaction product thus, described method comprises the following steps:
(a) provide the diphasic system of forming by the organic solvent of aqueous buffer and at least a the lipid epoxide that contains, particularly lipid acid epoxide or triglyceride level epoxide;
(b) diphasic system with excessive in a large number epoxide that described interfacial enzymes and step (a) is provided mixes, so that the nucleophilicity surface reaction base of enzyme, particularly amino, with epoxy reaction, make the enzyme (Fig. 1) that covalently is coated with lipid acid or is coated with triglyceride level;
(c) described carrier is joined in the mixture of step (b) and mix;
(d) from the mixture that step (c) is obtained, isolate the lipid-interfacial enzymes mixture that is fixed on the described carrier.
With before the enzyme biphasic solution is mixed, wash described carrier alternatively and desalt and organism to remove.
In this method of the present invention, insoluble carrier is by physical adsorption or by being covalently bonded in the functional group and can the bonding interface enzyme.Carrier is preferably porous support, it can be an organic or inorganic, preferably be selected from the group of forming by porous inorganic carrier (as silica-based or alumina type carrier) and organic carrier (as the polymer class carrier), wherein said carrier can contain alternatively just like epoxy group(ing) or aldehyde radical isoreactivity functional group, perhaps ionic group.
It is said, in the step of epoxidizing method of the present invention (a) used organic solvent can for but be not limited to alkanes (as octane), alcohols (as n-Octanol), aldehydes (as capraldehyde), ethers (as diisopropyl ether) or ketone (as methyln-hexyl ketone) and their any mixture.
Interfacial enzymes by method preparation of the present invention is preferably lipase, esterase or Phospholipid hydrolase.Concrete limiting examples is for being derived from antarctic candida (Candida antarctica), fold candida (Candida rugosa), rice black root Mucor (Rhizomucor miehei), pseudomonas (Pseudomonas sp.), snow-white head mold (Rhizopus niveus), mucor javanicus (Mucorjavanicus), rice black wool mould (Mucor miehei), Rhizopus oryzae (Rhizopus oryzae), aspergillus niger (Aspergillus niger), penicillium camembertii (Penicillium camembertii), Alcaligenes (Alcaligenes sp.), bulkholderia cepasea (Burkholderia sp.), dredge the thermophilic hyphomycete of continuous shape (Thermomyces lanuginosa), thickness look bacillus (Chromobacterium viscosum), the enzyme of Semen Fructus Chaenomelis and pancreatin.
The interfacial enzymes of the present invention that is fixed on the solid porous carrier is locked in its activity conformation, be modified by the molecule (combining with the surface functional group of enzyme) that covalently is coated with a large amount of described lipids, and be characterised in that for height endurability as hydrophilic substrates such as short chain alcohol and short chain fatty acids by its initial epoxide group.
In the epoxidizing method of the present invention used carrier by physical adsorption in functional group or can be in conjunction with the enzyme of described lipid lining by being covalently bonded in functional group, and can be organic carrier or inorganic carrier, preferably be selected from as inorganic carriers such as silica-based or alumina type carriers with as organic carriers such as polymer class carriers, and described carrier can contain ionic group and as epoxy group(ing) or aldehyde radical isoreactivity functional group, perhaps described carrier can be ion exchange resin.
In the preparation of the enzyme of this lipid lining of the present invention, lipid is preferred but be not limited to free fatty acids or fatty acid alkyl ester epoxide, sugar fatty acid ester epoxide, medium chain and chain alkyl glucosides, phosphatide epoxide, polyoxyethylene glycol epoxide derivate or quaternary ammonium salt epoxide.The common following acquisition of the epoxide of unsaturated lipids substrate: at least one two key is oxidized to epoxide group by chemistry or biochemical catalysis (for example in the presence of hydrogen peroxide, utilizing lipase).
In another embodiment, the present invention relates to a kind of preparation and be fixed in the method for the interfacial enzymes of the modification on the solid carrier, wherein, described enzyme is surrounded by hydrophobic microenvironment, can avoid inactivation and/or gathering in the presence of hydrophilic substrates and/or reaction product thus, said method comprising the steps of: system that (i) be made up of aqueous buffer or the diphasic system of (ii) being made up of aqueous buffer and organic solvent (a) are provided; (b) add hydrophobic polymer carrier to arbitrary described system (i) or (ii); (c) in the mixture that step (b) is obtained, add described interfacial enzymes and mixing; (d) from the mixture that step (c) is obtained, isolate the interfacial enzymes that is fixed on the described hydrophobic carrier.
In this method, described organic solvent can for but be not limited to octane, octane-iso, normal hexane, n-Octanol Di Iso Propyl Ether and oils triglyceride level.Hydrophobic polymer carrier can for but be not limited to hydrophobic aliphatic acrylic acid or the like cross-linked polymer or hydrophobicity aromatic series cross-linked polymer, the example is respectively Amberlite
RXAD 7HP and Amberlite
RXAD 1600.
In all methods of the present invention, enzyme can be a lipase, esterase or Phospholipid hydrolase, for example antarctic candida (Candida antarctica), fold candida (Candida rugosa), rice black root Mucor (Rhizomucor miehei), rice black wool mould (Mucor miehei), pseudomonas (Pseudomonas sp.), snow-white head mold (Rhizopus niveus), mucor javanicus (Mucorjavanicus), Rhizopus oryzae (Rhizopus oryzae), aspergillus niger (Aspergillus niger), penicillium camembertii (Penicillium camembertii), Alcaligenes (Alcaligenes sp.), bulkholderia cepasea (Burkholderia sp.), dredge the thermophilic hyphomycete of continuous shape (Thermomyces lanuginosa), thickness look bacillus (Chromobacterium viscosum), Semen Fructus Chaenomelis and pancreatin.
The immobilization hydrophobization enzyme of the immobilization hydrophobization enzyme of modification of the present invention or the modification by method of the present invention preparation can be advantageously used in the manufacturing fatty acid alkyl ester, and described fatty acid alkyl ester can be used as biofuel or as surface active ingredient preparation intermediate.
Thus, this paper provides the enzymatic means for preparing structurized lipid, said method comprising the steps of: in the presence of the immobilized enzyme of the immobilized enzyme of modification of the present invention or the modification by method preparation of the present invention, will as fatty acid sources such as free fatty acids, triglyceride level, fatty acid ester, partial glyceride, phosphatide or other derivative of fatty acid with as pure reaction the such as methyl alcohol.
In another embodiment; the present invention relates to the method for preparing lipid acid short-chain alkyl ester, be preferably fatty acid methyl ester (biofuel); described method comprises in the fatty acid source that mixture obtained that progressively methyl alcohol is joined by the vegetables oil, animal oil, algal oil or the fish oil that contain lipase of the present invention or the lipase by method of the present invention preparation or at least two kinds of above-mentioned oil; and reaction is carried out under proper condition, be converted into fatty acid methyl ester up to described fatty acyl group or lipid acid.In this method, vegetables oil can for but be not limited to soybean oil, Canola oil, rapeseed oil, sweet oil, Viscotrol C, plam oil, sunflower seed oil, peanut oil, cotton seed oil, leprosy fruit oil, useless cooking oil or be derived from any oils triglyceride level in non-edible plant source.
With reference to the following drawings the present invention is described in more detail below.
Description of drawings
Fig. 1: the schematically illustrating of method of using lipid acid epoxide covalency lining enzyme.
Fig. 2 A: in buffered soln, the contrast of the hydrolytic activity of the hydrolytic activity of immobilization rice black wool of the present invention mould (M.miehei) lipase and the same enzyme of immobilization and tensio-active agent or the non-covalent lining of lipid.
Fig. 2 B: in buffered soln, the contrast of the hydrolytic activity of the same enzyme of the composite reactive of immobilization M.miehei lipase of the present invention and immobilization and tensio-active agent or the non-covalent lining of lipid.
Fig. 3 A: in acetone, the contrast of the hydrolytic activity of the same enzyme of the hydrolytic activity of immobilization M.miehei lipase of the present invention and immobilization and tensio-active agent or the non-covalent lining of lipid.
Fig. 3 B: in acetone, the contrast of the hydrolytic activity of the same enzyme of the composite reactive of immobilization M.miehei lipase of the present invention and immobilization and tensio-active agent or the non-covalent lining of lipid.
Fig. 4 A: in normal hexane, the contrast of the hydrolytic activity of the same enzyme of the hydrolytic activity of immobilization M.miehei lipase of the present invention and immobilization and tensio-active agent or the non-covalent lining of lipid.
Fig. 4 B: in normal hexane, the contrast of the hydrolytic activity of the same enzyme of the composite reactive of immobilization M.miehei lipase of the present invention and immobilization and tensio-active agent or the non-covalent lining of lipid.
Fig. 5: in the successive type transesterification reaction, utilize the reaction conversion ratio (%) that is converted into fatty acid methyl ester when being fixed in use with the thermophilic hyphomycete of thin continuous shape (Thermomyces lanuginosa) lipase on the various matrix of a collection of biological catalyst.Reaction conditions: soybean oil (2.5g), methyl alcohol (0.3g is divided into three parts of intervals and added in 1 hour) and immobilized lipase (250mg) were mixed 4 hours in the constant temperature wobbler at 30 ℃.
Fig. 6: in the successive type transesterification reaction, utilize the reaction conversion ratio (%) that is converted into fatty acid methyl ester when being fixed in use with the antarctic candida B on the various matrix of a collection of biological catalyst (Candida Antarctica B) lipase.Reaction conditions as shown in Figure 5.
Fig. 7: in the successive type transesterification reaction, utilize the reaction conversion ratio (%) that is converted into fatty acid methyl ester when being fixed in use with the pseudomonas cepacia on the various matrix of a collection of biological catalyst (Pseudomonas cepacia) lipase.Reaction conditions as shown in Figure 5.
Fig. 8: in the successive type transesterification reaction, utilize the reaction conversion ratio (%) that is converted into fatty acid methyl ester when being fixed in use with the Alcaligenes on the various matrix of a collection of biological catalyst (Alcaligenes sp.) lipase.Reaction conditions as shown in Figure 5.
Embodiment
A kind ofly to be used to prepare high activity and stable immobilized interfacial enzymes in order searching, particularly to have the novel method of the lipase and the Phospholipid hydrolase of height endurability for hydrophilic substrates (as short chain alcohol and short chain fatty acid), the inventor finds, near the hydrophobic microenvironment of the transesterification reaction medium the reactive site of enzyme can be used as the activity that strengthens lipase and improves its means to the tolerance of other hydrophilic agent that may exist in wetting ability short chain alcohol and acid and the reaction mixture.
The inventor develops different enzyme preparations thus, and wherein enzyme is fixed on the insoluble matrix and has been endowed hydrophobicity.As follows, described enzyme both can for example directly be endowed hydrophobicity by adhering to lipotropy residue (for example using lipid epoxide epoxidation), also can be endowed hydrophobicity on the hydrophobic base by being fixed in, described hydrophobic base provides the hydrophobic microenvironment of surrounding enzyme or imbedding enzyme.The present invention proves that near the hydrophobic microenvironment the reactive site of enzyme has played the effect of buffer zone, and described buffer zone can prevent that enzyme is exposed to the substrate and the product of the possess hydrophilic property part of inhibition concentration.The short chain alcohol that the hydrophobic microenvironment that provides for enzyme is responsible for controlling non-inhibition concentration scope enters the reactive site of enzyme, and near the wetting ability reaction product of being responsible for forming the reactive site with enzyme is removed to reaction medium.
Therefore, according to an aspect of the present invention, the hydrophobicity immobilized enzyme can be by the technology preparation of two steps, and described technology is as follows basically:
Step 1:, force all interfacial enzymes molecules to adopt its activity conformation (referring to Fig. 1) by with the lipid epoxide reaction of interfacial enzymes molecule with the diphasic system of forming by water and the organic phase that contains the lipid epoxide.The lipid epoxide excessively exists in a large number, so each enzyme molecule all covalently is coated with a large amount of lipid molecules.
Step 2: suitable carriers is joined in the diphasic system of the enzyme that contains described covalency lining.
Under these conditions, be positioned at the two-phase enzyme molecule that covalently is coated with lipid residue or mixture at the interface, by simple physical absorption, with the activated resin covalent attachment that contains functional group's (as epoxy group(ing) or aldehyde radical etc.) or by the absorption on ion exchange resin, can be fixed to easily on the described carrier.
In the preparation of the immobilized interfacial enzymes of active modification of the present invention, adopted this two steps technology.
According to this on the one hand, the present invention relates to a kind of method of immobilized interfacial enzymes, particularly lipase, esterase and Phospholipid hydrolase of the modification for preparing stable, high activity, wherein, provide the diphasic system of forming by aqueous buffer and at least a organic solvent that contains the lipid epoxide; Described interfacial enzymes is mixed with a large amount of excessive described diphasic systems; Solid carrier is joined in the described mixture; And isolate the interfacial enzymes of the lipid covalency lining of being fixed on the described carrier.
Solid carrier is preferably porous support, it can be an organic or inorganic, particularly be selected from the group of forming by porous inorganic carrier (as silica-based or alumina type carrier) and organic carrier (as the polymer class carrier), wherein said carrier can contain alternatively just like epoxy group(ing) or aldehyde radical isoreactivity functional group, perhaps ionic group.Some concrete carriers have been provided in the following examples, the particularly table 1.
Diphasic system is by suitable aqueous buffer and organic solvent preparation.Organic solvent can for but be not limited to alkanes (as octane), ethers (as Di Iso Propyl Ether), alcohols (as n-Octanol), aldehydes (as capraldehyde), ketone (as methyln-hexyl ketone) and their any mixture.
Immobilized enzyme of the present invention or the immobilized enzyme by above-mentioned epoxidizing method preparation of the present invention have activity, stable especially and to having height endurability as hydrophilic substrates such as short chain alcohol and short chain fatty acids very much.Even after 10 secondary responses circulations, still keep 90% the activity of having an appointment.This stability has great economic implications.
On the other hand, the present invention relates to the alternative approach of lipase hydrophobization, described method has been avoided the needs to epoxidation and use lipid part lining enzyme.Physical attachment in the immobilized enzyme of hydrophobic microenvironment by hydrophobic porous polymeric matrix is contacted and makes with the aqueous solution or with the water that contains different lipase-organic solvent bilayer [also being called two-phase] system.Be used for making the oils triglyceride level of biofuel and glycerine and the immobilized enzyme that the test of the transesterification reaction between the methyl alcohol is so made, this transesterification reaction is as the reaction model among the present invention.Be not subjected to the restriction of any theory, the result shows that the hydrophobicity of microenvironment can cause near the concentration of the hydroaropic substance the enzyme molecule to reduce.These hydroaropic substances may be employed substrates in the reaction, also may be the formed products of reaction.This " hydrophobization " biological catalyst has guaranteed to arrive near the wetting ability short chain alcohol of enzyme and the concentration of acid reaction is controlled, and/or can remove near any hydroaropic substance that forms the enzyme molecule rapidly.As main result, in case reach the wetting ability product of removing reaction formation fast by near the concentration of reactants the control arrival immobilized enzyme, the enzyme molecule is avoided the influence of hydrophobic substrate and product.Use four kinds of different lipase that above-mentioned saying is tested, wherein, every kind of lipase all is fixed on the different carrier of four kinds of hydrophobicitys, shown in embodiment 5 independently.
In another embodiment, the present invention relates to prepare the method for lipid acid short-chain alkyl ester, particularly fatty acid methyl ester (biofuel).Generally speaking, in the method, at first progressively methyl alcohol is joined as vegetables oil, animal oil, algal oil, fish oil or be derived from any oil of fungi or the fatty acid sources such as mixture of at least two kinds of above-mentioned oil in.The lipase (preparing by for example method of the present invention) or the lipase that is fixed on the hydrophobic base of the present invention that will be fixed in the modification on the solid carrier that covalently is coated with lipid join in methyl alcohol/fatty acid source mixture, and reaction is carried out, be converted into fatty acid methyl ester until fatty acid source.
In the application's context, term " carrier ", " matrix ", " sorbent material " synonym are used interchangeably.
As disclosed herein and the explanation, should be appreciated that to the invention is not restricted to specific example disclosed herein, method steps and material, because these method stepss and material can change to a certain extent.Should also be appreciated that term used herein is only used for illustrating the purpose of embodiment rather than is intended to restriction, because scope of the present invention only is subjected to the restriction of claims and equivalent thereof.
Must be noted that as this specification sheets and claims usedly, what " a ", " an " and singulatives such as " the " comprised plural number refers to thing, unless offer some clarification in addition in the content.
In whole specification sheets and appended claim book, unless context has regulation in addition, otherwise word " comprises " and " comprising " will be understood that to mean the group that comprises illustrated integral body or step or a plurality of integral body or a plurality of steps, but do not get rid of any other integral body or the group of step or a plurality of integral body or a plurality of steps.
The representative of the following example technology that to be the inventor adopt when implementing each side of the present invention.Although it should be understood that these technology is the examples that are used for the preferred implementation of enforcement of the present invention, yet those skilled in the art will appreciate that according to present disclosure can carry out various modifications under the situations that does not deviate from desired extent of the present invention.
Embodiment
The preparation of immobilized lipase (Lipozyme TL)
To be derived from lipase (the Lipozyme TL 100L of 1ml that dredges the thermophilic hyphomycete of continuous shape (Thermomyces lanuginosa), Novozymes, Denmark) be mixed into by 1ml phosphate buffer (0.05M, pH 6.5) and 10ml and contain in the solvent pairs system that the normal hexane of lipid epoxide forms.Stirred this mixture 48 hours.Add carrier (1g) in this system and stirred this mixture 8 hours.Leach the carrier of the immobilized enzyme that contains modification and in moisture eliminator dried overnight, thereby obtain the immobilized lipase of the lipid covalency lining of high activity.
Table 1 has shown the relative transesterify activity of the Lipozyme TL 100L that is fixed on the different carriers.Reaction is undertaken by immobilized lipase (0.2g) is joined in soybean oil (2.5g) and the methyl alcohol (0.3g).Reaction system is mixed 30 ℃ of magnetic agitation or by jolting.React the fatty acid methyl ester that generates after 1 hour under these conditions and determine speed of reaction by measuring.
Table 1: the speed of reaction of the transesterify that the Lipozyme of different lipid acid epoxide modifications is used for the soybean oil triglyceride level when obtaining fatty acid methyl ester (FAME).Reaction conditions: sweet oil (2.5g) and methyl alcohol (0.2g) were mixed 1 hour with the lipase TL 100L (0.2g) that is fixed on the different carriers of modification.With reaction mixture with 300rpm 30 ℃ of joltings.
| Bearer type | Speed of reaction (micromole FAME/ minute gram biological catalyst) |
| ??Amberlite?XAD?4(Rohm&Haas，USA) | ??16 |
| ??Amberlite?XAD?16(Rohm&Haas) | ??12 |
| ??Amberlite?XAD?7HP(Rohm&Haas) | ??10 |
| ??Amberlite?XAD?16HP(Rohm&Haas) | ??22 |
| ??Duolite?XAD?761(Rohm&Haas) | ??11 |
| ??Amberlite?XAD?1180(Rohm&Haas) | ??9 |
| ??Amberlite?XAD?1600(Rohm&Haas) | ??8 |
| ??Duolite?A7(Rohm&Haas) | ??12 |
| ??Duolite?A561(Rohm&Haas) | ??10 |
| ??Duolite?A568(Rohm&Haas) | ??12 |
| Bearer type | Speed of reaction (micromole FAME/ minute gram biological catalyst) |
| ??Duolite?C467(Rohm&Haas) | ??6 |
| ??Amberlyst?A-21(Rohm&Haas) | ??10 |
| ??Dowex?monosphere?77(DOW，USA) | ??13 |
| ??Dowex?optipore?L493(DOW，USA) | ??12 |
| ??Dow?styrene?DVB(DOW，USA) | ??18 |
| ??MTO?Dowex?optipore?SD-2(DOW，USA) | ??10 |
| ??Dowex?MAC-3 | ??9 |
| ??Amberlite?FPA53(Rohm&Haas) | ??11 |
| ??Amberlite?FPC22H(Rohm&Haas) | ??10 |
| ??Amberlite?FPA40C1(Rohm&Haas) | ??2 |
| ??AmberliteIRC50(Rohm&Haas) | ??7 |
| ??Purolire?A109(Purolite，USA) | ??12 |
The preparation of immobilized lipase
Use different lipase (100mg) and use the triglyceride level epoxide to repeat the immobilization program of embodiment 1.The speed of reaction that generates fatty acid methyl ester under the above-mentioned condition is as shown in table 2.
Table 2: the speed of reaction of the transesterify that the lipase of different lipid acid epoxide modifications is used for the soybean oil triglyceride level when obtaining fatty acid methyl ester (FAME).Reaction conditions: soybean oil (2.5g) and methyl alcohol (0.3g) were mixed 1 hour with the different lipase (0.2g) that covalently is coated with the triglyceride level epoxide and is fixed on the Amberlite XAD 4.With reaction mixture with 300rpm 30 ℃ of joltings.
| Bearer type | Speed of reaction (micromole FAME/ minute gram biological catalyst) |
| Antarctic candida (Candida antarctica) | ??9.6 |
| Bearer type | Speed of reaction (micromole FAME/ minute gram biological catalyst) |
| Fold candida (Candida rugosa) | ??7.7 |
| Rice black root Mucor (Rhizomucor miehei) | ??12.5 |
| Pseudomonas cepacia (Pseudomonas cepacia) | ??16.2 |
| Penicillium camembertii (Penicillium camembertii) | ??4.2 |
| Alcaligenes (Alcaligenes sp.) | ??12.3 |
| Snow-white head mold (Rhizopus niveus) | ??3.4 |
| Mucor javanicus (Mucor javanicus) | ??12.3 |
| Rhizopus oryzae (Rhizopus oryzae) | ??14.2 |
| Aspergillus niger (Aspergillus niger) | ??6.3 |
| Bulkholderia cepasea (Burkholderia sp.) | ??12.1 |
| Dredge the thermophilic hyphomycete of continuous shape (Thermomyces lanuginosa) | ??17.1 |
| Thickness look bacillus (Chromobacterium viscosum) | ??15.1 |
Embodiment 3
Be used to prepare the immobilized lipase of fatty acid methyl ester (biofuel)
Use Amberlite XAD 8 or Celite (diatomite, powder) as carrier, buffer reagent (contrast) normal hexane or acetone (Ac) prepare the M.miehei lipase goods of immobilization modification as organic solvent according to the program of embodiment 1.
These goods are used for the preparation of fatty acid methyl ester (biofuel).By adding immobilized lipase (100mg) and 30 ℃ of jolting reaction mediums 6 hours and initiation reaction.
The hydrolytic activity and the composite reactive of different enzyme preparation [enzyme covalently is coated with buffer reagent-epoxide-oil that oleic buffer reagent-epoxide-OA and enzyme covalently are coated with oil (triglyceride level, for example triolein)] are compared with the activity of following other enzyme preparation (covalently not being coated with lipid): enzyme is fixed in the buffer reagent (contrast) on the carrier simply; Carrier immobilized enzyme is coated with buffer reagent-oleic acid (buffer reagent-OA) of lipid acid; Carrier immobilized enzyme is coated with the buffer reagent-SMO of Arlacel-60.
The result is as shown in Fig. 2～Fig. 4.Viewed as us, the hydrolytic activity of the enzyme that covalently is coated with lipid composition of the present invention and the activity that composite reactive is higher than other enzyme preparation quite significantly.
In addition, even after the reaction cycle more than 10 times, still kept most of active (data not shown goes out) of enzyme.
Therefore, the enzyme preparation of epoxidation modification of the present invention shows the stability of high reactivity and raising.
The immobilization of lipase in buffer reagent or diphasic system
At room temperature [3000 units dredge the thermophilic hyphomycete of continuous shape (Thermomyceslanuginosa) lipase, (the two is all from Novozymes for antarctic candida (Candida antarctica) lipase B with lipase, Denmark), pseudomonas cepacia (Pseudomonas cepacia) lipase (AmanoEnzyme Inc., Japanese) or Alcaligenes (Alcaligenes sp.) lipase (Meito Sangyo, Japan)] in the buffered soln that contains polymer support (1g) (10ml, pH=7) the middle mixing 8 hours.Leach immobilized enzyme and dry on silicon-dioxide in moisture eliminator.Carrying out identical program by in the buffered soln of equal volume and the diphasic system that organic solvent (for example octane-iso) is formed.
Used all by U.S. Rohm﹠amp; The following carrier that Haas makes:
-Amberlite XAD 1600 is defined as hydrophobic adsorbent;
-Amberlite XAD761 is defined as the wetting ability sorbent material;
-Amberlite XAD7HP is defined as polarity and non-polar adsorbent; With
-Amberlite IRA-958 is defined as polarity anionite-exchange resin.
The application of the immobilized lipase of embodiment 4 in biofuel is made
Utilize immobilized lipase that the transesterify of oils triglyceride level and methyl alcohol estimates preparation among the embodiment 4 in biofuel and as the activity in the manufacturing of the glycerine of by product.By to magnetic agitation contain methyl alcohol (0.3g, be divided into 3 parts in 4 hours every interval 1 hour add 1 part) soybean oil (2.5g) in add 10 weight % immobilized lipase come initiation reaction.
Shown in Fig. 5～8 using and after 4 hours, during the permutoid reaction medium, be fixed in the transformation efficiency (%) that oils is converted into fatty acid methyl ester of the different lipase of different substrates simultaneously with a collection of biological catalyst.But enzyme that be fixed in other type of carrier on identical with other compared, and is fixed in as Amberlite
RLipase on XAD 1600 hydrophobic carriers such as grade (sorbent material) has kept its transesterify activity in the application repeatedly of much more lot number.In addition, can also be clear that for the reaction of this class, all are fixed in lipase on the hydrophilic carrier, and to have shown relatively poor transesterify applicability repeatedly active and continuous batch relatively poor.Be also noted that and be fixed in as Amberlite
RXAD761 and Amberlite
RLipase on the hydrophilic carriers such as IRA-958 has formed the aggregate of biological catalyst, has the formed wetting ability product of saturated reaction on it, i.e. glycerine.Because the accumulation of the product that forms, owing near the methyl alcohol of the high density immobilized enzyme, biological catalyst shows lower activity and less number of applications repeatedly simultaneously.
On the contrary, be fixed in as Amberlite
RXAD 1600 and Amberlite
RLipase on the hydrophobic carriers such as XAD7HP, the particularly thin thermophilic hyphomycete of continuous shape (Thermomyces lanuginosa) lipase and pseudomonas cepacia (Pseudomonas cepacia) lipase have obtained higher transesterify activity, and have kept its transesterify activity when using with a collection of biological catalyst in surpassing 50 times circulation repeatedly.
Claims (20)
1. interfacial enzymes that is fixed in the modification on the solid carrier, wherein, described enzyme is surrounded by hydrophobic microenvironment, avoids inactivation and/or gathering in the presence of hydrophilic agent, substrate and/or reaction product thus.
2. the interfacial enzymes of modification as claimed in claim 1, wherein, described enzyme covalently is coated with lipid groups.
3. the interfacial enzymes of modification as claimed in claim 2, wherein, described carrier is by absorption or by being covalently bonded in the functional group and can be in conjunction with described enzyme.
4. as the interfacial enzymes of each described modification of claim 2 and 3, wherein, described carrier is organic or inorganic, preferably be selected from the group of forming by inorganic carrier as silica-based or alumina type carrier and the organic carrier as the polymer class carrier, wherein said carrier can contain ionic group and the active function groups as epoxy group(ing) or aldehyde radical, and perhaps described carrier is an ion exchange resin.
5. as the interfacial enzymes of each described modification of claim 2～4, wherein, described lipid epoxide is selected from lipid acid, fatty acid alkyl ester, sugar fatty acid ester, medium chain and chain alkyl glucosides, phosphatide, polyethyleneglycol derivative and quaternary ammonium salt.
6. the interfacial enzymes of modification as claimed in claim 1, wherein, described solid carrier is the hydrophobic solid carrier.
7. as each described enzyme of claim 1～6, described enzyme is lipase, esterase or Phospholipid hydrolase.
8. enzyme as claimed in claim 7, wherein, described enzyme is selected from by antarctic candida (Candida antarctica), fold candida (Candida rugosa), rice black root Mucor (Rhizomucor miehei), pseudomonas (Pseudomonas sp.), snow-white head mold (Rhizopusniveus), rice black wool mould (Mucor miehei), mucor javanicus (Mucor javanicus), Rhizopus oryzae (Rhizopus oryzae), aspergillus niger (Aspergillus niger), penicillium camembertii (Penicilliumcamembertii), Alcaligenes (Alcaligenes sp.), bulkholderia cepasea (Burkholderiasp.), dredge the thermophilic hyphomycete of continuous shape (Thermomyces lanuginosa), thickness look bacillus (Chromobacterium viscosum), the group that Semen Fructus Chaenomelis and pancreatin are formed.
9. method that is used to prepare the interfacial enzymes that is fixed in the modification on the insoluble carrier said method comprising the steps of:
(a) provide the system of forming by aqueous buffer and at least a organic solvent that contains the lipid epoxide;
(b) described interfacial enzymes is mixed with the solvent pairs system that step (a) is provided;
(c) described carrier is joined in the mixture of step (b) and mix;
(d) from the mixture that step (c) is obtained, isolate the described interfacial enzymes that is fixed on the described carrier.
10. method as claimed in claim 9, wherein, described carrier is organic porous support or inorganic porous carrier, preferably be selected from the group of being made up of porous inorganic carrier as silica-based or alumina type carrier and the organic carrier as the polymer class carrier, wherein said carrier contains ionic group or the active function groups as epoxy group(ing) or aldehyde radical so alternatively.
11. each described method as claim 9 and 10, wherein, described organic solvent is selected from alkanes (as octane), ethers (as diisopropyl ether), alcohols (as n-Octanol), aldehydes (as capraldehyde) and ketone (as methyln-hexyl ketone) and their any mixture.
12. as each described method of claim 9～11, wherein, described lipid epoxide is selected from lipid acid, fatty acid methyl ester, sugar fatty acid ester, medium chain and chain alkyl glucosides, phosphatide, polyethyleneglycol derivative and quaternary ammonium salt.
13. a method that is used to prepare the interfacial enzymes that is fixed in the modification on the solid carrier, wherein, described enzyme is surrounded by hydrophobic microenvironment, avoids inactivation and/or gathering in the presence of hydrophilic substrates and/or reaction product thus, and described method comprises the following steps:
(a) system that (i) be made up of aqueous buffer or the diphasic system of (ii) being made up of aqueous buffer and organic solvent are provided;
(b) add hydrophobic polymer carrier to arbitrary described system (i) or (ii);
(c) in the mixture that step (b) is obtained, add described interfacial enzymes and mixing; With
(d) from the mixture that step (c) is obtained, isolate the described interfacial enzymes that is fixed on the described hydrophobic carrier.
14. method as claimed in claim 13, wherein, described organic solvent is selected from octane, octane-iso, normal hexane, n-Octanol Di Iso Propyl Ether and oils triglyceride level.
15. as each described method of claim 13 and 14, wherein, described hydrophobic polymer carrier is selected from the group of hydrophobic aliphatic acrylic acid or the like cross-linked polymer or hydrophobicity aromatic series cross-linked polymer, the two is respectively as Amberlite
RXAD 7HP and Amberlite
RXAD1600.
16. as each described method of claim 9～15, wherein, described enzyme is lipase, esterase or Phospholipid hydrolase.
17. method as claimed in claim 16, wherein, described enzyme is selected from by antarctic candida (Candida antarctica), fold candida (Candida rugosa), rice black root Mucor (Rhizomucor miehei), rice black wool mould (Mucor miehei), pseudomonas Pseudomonassp.), snow-white head mold (Rhizopus niveus), mucor javanicus (Mucor javanicus), Rhizopus oryzae (Rhizopus oryzae), aspergillus niger (Aspergillus niger), penicillium camembertii (Penicilliumcamembertii), Alcaligenes (Alcaligenes sp.), bulkholderia cepasea (Burkholderiasp.), dredge the thermophilic hyphomycete of continuous shape (Thermomyces lanuginosa), thickness look bacillus (Chromobacterium viscosum), the group that Semen Fructus Chaenomelis and pancreatin are formed.
18. immobilized enzyme as each described modification of claim 1～8, perhaps by as the immobilized enzyme of the modification for preparing of the described method of each of claim 9～17, it is used to make fatty acid alkyl ester, and described fatty acid alkyl ester is as biofuel or as surface active ingredient preparation intermediate.
19. one kind is used to prepare lipid acid short-chain alkyl ester, the method of preferred fatty acid methyl esters (biofuel), said method comprising the steps of: progressively methyl alcohol is joined vegetables oil, animal oil, in algal oil or fish oil or at least two kinds of these the oily mixtures, described vegetables oil, animal oil, algal oil or fish oil or at least two kinds of these oily mixtures contain the lipase of each described lipase of claim 1～8 or each the described method preparation by claim 9～17, and reaction is carried out under proper condition, be converted into fatty acid methyl ester up to described oils triglyceride level.
20. method as claimed in claim 19, wherein, described vegetables oil is soybean oil, Canola oil, rapeseed oil, sweet oil, plam oil, sunflower seed oil, peanut oil, cotton seed oil, useless cooking oil or any oils triglyceride level that is derived from the non-edible plant source.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| IL183084A IL183084A0 (en) | 2007-05-09 | 2007-05-09 | Modified-immobilized enzymes of high tolerance to hydrophilic substrates in organic media |
| IL183084 | 2007-05-09 | ||
| PCT/IL2008/000631 WO2008139455A2 (en) | 2007-05-09 | 2008-05-07 | Modified-immobilized enzymes of high tolerance to hydrophilic substrates in organic media |
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| CN101790582A true CN101790582A (en) | 2010-07-28 |
| CN101790582B CN101790582B (en) | 2015-01-21 |
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| US (1) | US9068175B2 (en) |
| EP (1) | EP2152866B1 (en) |
| JP (1) | JP5484318B2 (en) |
| KR (1) | KR101560842B1 (en) |
| CN (1) | CN101790582B (en) |
| AU (1) | AU2008249564B2 (en) |
| BR (1) | BRPI0811312A2 (en) |
| CA (1) | CA2686917C (en) |
| DK (1) | DK2152866T3 (en) |
| ES (1) | ES2447540T3 (en) |
| IL (2) | IL183084A0 (en) |
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| WO (1) | WO2008139455A2 (en) |
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| CN104293764A (en) * | 2013-07-19 | 2015-01-21 | 丰益(上海)生物技术研发中心有限公司 | Phosphatidase A1 immobilization method |
| CN106811455A (en) * | 2015-11-27 | 2017-06-09 | 丰益(上海)生物技术研发中心有限公司 | Immobilised enzymes and preparation method thereof |
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| US9168469B2 (en) | 2004-12-22 | 2015-10-27 | Chemtor, Lp | Method and system for production of a chemical commodity using a fiber conduit reactor |
| IL180598A0 (en) * | 2007-01-08 | 2007-07-04 | Basheer Sobhi | Immobilized interfacial enzymes of improved and stabilized activity |
| US8617866B2 (en) * | 2007-11-28 | 2013-12-31 | Sobhi Basheer | Robust multi-enzyme preparation for the synthesis of fatty acid alkyl esters |
| AR080117A1 (en) * | 2010-03-01 | 2012-03-14 | Trans Bio Diesel Ltd | A PROCESS FOR THE ENZYMATIC SYNTHESIS OF ALKYL ESTERS OF FATTY ACIDS |
| US10000731B2 (en) | 2010-03-01 | 2018-06-19 | Trans Bio-Diesel Ltd. | Enzymatic transesterification/esterification processes employing lipases immobilized on hydrophobic resins in the presence of water solutions |
| JP5178888B2 (en) * | 2011-08-02 | 2013-04-10 | 日清オイリオグループ株式会社 | Method for producing transesterified oil |
| CA2842032C (en) * | 2011-08-31 | 2016-09-27 | Trans Bio-Diesel Ltd. | Enzymatic transesterification with lipases immobilized on hydrophobic resins in water solutions |
| CA3109603C (en) | 2012-09-18 | 2022-09-27 | Chemtor, Lp | Use of a fiber conduit contactor for metal and/or metalloid extraction |
| WO2014100454A1 (en) * | 2012-12-19 | 2014-06-26 | John Lee Massingill | Enzymatic chemical processing in a fiber conduit apparatus |
| US20150353970A1 (en) | 2012-12-31 | 2015-12-10 | Trans Bio-Diesel Ltd. | Enzymatic transesterification/esterification processing systems and processes employing lipases immobilzed on hydrophobic resins |
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| CN104293764A (en) * | 2013-07-19 | 2015-01-21 | 丰益(上海)生物技术研发中心有限公司 | Phosphatidase A1 immobilization method |
| CN103451174A (en) * | 2013-09-22 | 2013-12-18 | 清华大学 | Enzyme-macromolecule conjugate and preparing method and application thereof |
| CN106811455A (en) * | 2015-11-27 | 2017-06-09 | 丰益(上海)生物技术研发中心有限公司 | Immobilised enzymes and preparation method thereof |
Also Published As
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|---|---|
| BRPI0811312A2 (en) | 2014-12-30 |
| CA2686917C (en) | 2013-07-09 |
| MX2009012132A (en) | 2009-12-01 |
| US20100209982A1 (en) | 2010-08-19 |
| EP2152866A2 (en) | 2010-02-17 |
| ES2447540T3 (en) | 2014-03-12 |
| EP2152866A4 (en) | 2010-09-01 |
| IL202010A0 (en) | 2011-08-01 |
| JP5484318B2 (en) | 2014-05-07 |
| WO2008139455A2 (en) | 2008-11-20 |
| US9068175B2 (en) | 2015-06-30 |
| WO2008139455A3 (en) | 2010-02-25 |
| AU2008249564A1 (en) | 2008-11-20 |
| CN101790582B (en) | 2015-01-21 |
| KR20100023853A (en) | 2010-03-04 |
| IL202010A (en) | 2016-02-29 |
| DK2152866T3 (en) | 2014-03-17 |
| AU2008249564B2 (en) | 2014-05-22 |
| CA2686917A1 (en) | 2008-11-20 |
| IL183084A0 (en) | 2007-09-20 |
| KR101560842B1 (en) | 2015-11-20 |
| EP2152866B1 (en) | 2013-12-18 |
| JP2010525834A (en) | 2010-07-29 |
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